1. Field of the Invention
The invention is directed to peptides as adjuvants in vaccine production and methods of using peptides to increase the immune response to vaccine formulations. In particular, the invention is directed to methods of producing vaccines having smaller dosing of an antigenic material and/or greater immune response while maintaining a high level of efficacy.
2. Description of the Background
Vaccines have been used for the last 216 years to help prevent the spread of infectious diseases within a population of people. Since Edward Jenner's 1796 discovery that cowpox material created human immunity to smallpox, advances in awareness and technologies have seen the practice of vaccination become commonplace for a wide variety of infectious agents. Over the last fifty years, vaccination has transformed lives in both developed and undeveloped countries alike, contributing to the eradication of smallpox and large reductions of previously common diseases such as measles, typhoid and polio.
While advances in vaccinations have prevented an uncountable number of deaths from disease, it is estimated that 1.2 million people die needlessly each year because they do not receive vaccinations for tetanus, whooping cough, measles, tuberculosis, diphtheria and polio-vaccinations that have been widely available in developed countries since at least the 1960's. At least an additional 2 million deaths per year could be prevented by the full utilization of vaccines against Haemophilus influenzae type B (Hib) and hepatitis B.
The discrepancies of the use of existing vaccines can be attributed to the fact that many existing vaccines have not been affordable for developing countries. More than ten years after the universal introduction of the Hib vaccine, which protects against ear infections, meningitis, pneumonia and sepsis, fewer than 10% of children in the poorest 75% of countries were regularly receiving it as part of their immunization package due to the inability of governments and/or health care infrastructures to pay for the per dose immunization cost.
Between 1980 and 2009, the world's population more than doubled. However, during that time, the number of diphtheria cases dropped by 99%, polio cases by 97% and measles and pertussis cases by 95% each. These sharp decreases coincided with the widespread introduction of affordable vaccines into the marketplace. By increasing the affordability and access to vaccines and immunizations throughout the developed and underdeveloped world, within the next 20 years these diseases can be effectively eradicated.
In the case of poliomyelitis (polio); a highly infectious incurable viral disease that attacks the nervous system and causes permanent paralysis, prevention through vaccination is the only protection for a population. There are currently two types of polio vaccines available. Oral polio vaccine (OPV) and inactivated polio vaccine (IPV), delivered through injection. Although oral polio vaccine has been a valuable tool against the spread of the disease and is very inexpensive ($0.12-$0.20 per dose), OPV has two main drawbacks: the attenuated virus in OPV can mutate into a vaccine derived poliovirus (known as VDVP) that can cause polio infections in a population and on other, rare occasions, can result in vaccine-associated paralytic polio. On the other hand, currently available inactivated polio vaccines are more safe and effective, but cost nearly $3 per dose; too much for many underdeveloped countries to afford.
As vaccination will be needed for many years and perhaps decades after the spread of polio is eradicated, and since VDPV's will continue to occur until all populations switch to IPV, it is imperative that the price of IPV's are reduced so that underdeveloped countries can afford their widespread use. Several strategies have been proposed including the use of adjuvants to increase the body's immune response and/or needle-free delivery mechanisms that require smaller amounts of antigenic material per dose.
Each day, the human body is attacked by bacteria, viruses or other infectious agents. When a person becomes infected with a disease causing agent, the body's built-in immune system attempts to defend against the foreign agent. When the body successfully defends itself, immunity against the infectious agent is the result. When the body's natural defenses fail to quell the attack, an infection often results. In the natural process of developing immunity, B cells produced by the body produce substances known as antibodies that act against the specific infectious agent and create a “log” of this experience that can be called upon for protection when exposed to the same infectious agent again months, years or even decades later. Any subsequent time the person encounters that specific infectious agent; the circulating antibodies quickly recognize it and enable it to be eliminated from the body by other immune cells before signs of disease develop. It has been estimated that antibodies which can recognize as many as 10,000 different antigens or foreign infectious agents are circulating the blood stream.
A vaccine works in a similar way in that an antigenic response is produced. However, instead of initially suffering the natural infection and risking illness in order to develop this protective immunity, vaccines create a similar protective immunity without generally exposing the body to a condition wherein an infection could occur.
Development of vaccines against both bacterial and viral diseases has been one of the major accomplishments in medicine over the past century. While effective vaccines have been developed for a large number of diseases, the need for development of safe and effective vaccines for a number of other diseases remain.
Several basic strategies are used to make vaccines. One strategy is directed toward preventing viral diseases by weakening or attenuating a virus so that the virus reproduces very poorly once inside the body. Measles, mumps, rubella (German measles) and chickenpox (varicella) vaccines are made this way. Whereas natural viruses usually cause disease by reproducing themselves many thousands of times, weakened vaccine viruses reproduce themselves approximately 20 times. Such a low rate of replication is generally not enough to cause disease. Although the preparation of live, attenuated infectious agents as vaccines will often provide improved immunologic reactivity, such methods do increase the risk that the vaccine itself will be the cause of infection, and that the attenuated organism will propagate and provide a reservoir for future infection. One or two doses of live “weakened” viruses may provide immunity that is life-long; however, such vaccines cannot be given to people with weakened immune systems.
Another way to make viral vaccines is to inactivate the virus. By this method, viruses are completely inactivated or killed using a chemical. Killing the virus makes the virus unable to replicate in a body and cause disease. Polio, hepatitis A, influenza and rabies vaccines are made this way. The use of inactivated or killed bacterial or viral agents as a vaccine used to induce an antigenic response, although generally safe, will not always be effective if the antigenic characteristics of the agent are altered. An inactive virus can be given to people with weakened immune systems, but must be given multiple times to achieve immunity.
Vaccines may also be made using parts of a virus or bacteria. With this strategy, a portion of the virus is removed and used as a vaccine. The body is able to recognize the whole virus based on initial exposure to a portion of the virus. The hepatitis B vaccine for example, is composed of a peptide that resides on the surface of the hepatitis B virus.
Thus, one must generally choose between improved effectiveness and greater degree of safety when selecting between the inactivation and attenuation techniques for vaccine preparation. The choice is particularly difficult when the infectious agent is resistant to inactivation and requires highly rigorous inactivation conditions which are likely to degrade the antigenic characteristics which help to induce an immune response and provide subsequent immunity.
In addition to the dead or weakened infectious agent, vaccines usually contain sterile water or saline. Some vaccines are prepared with a preservative or antibiotic to prevent bacterial growth. Vaccines may also be prepared with stabilizers to help the vaccine maintain its effectiveness during storage. Other components may include an adjuvant which helps stimulate the production of antibodies against the vaccine to make it more effective.
Methods to prepare vaccines today involve treating samples with glutaraldehyde or formaldehyde to fix or cross-link the cells or infectious particles. Such treatments generally involve denaturation of the native forms of the infectious particles. A disadvantage to this approach is that the peptide coats of the infectious particles are damaged by this process, and thus may not be recognized by the immune system.
Many of the recent vaccine candidates are based on protected antigens, which are inherently less antigenic than the whole cell inactivated or live attenuated vaccines that were developed in the past. The challenge of formulating vaccines using these protected antigens is ensuring a sufficient immune response in vivo to convey immunity to the desired disease state. One manner in which this can be achieved is the discovery and development of novel adjuvants.
The goal of vaccination is to generate a strong immune response to the administered antigen, one that enables long-term protection against infection. This immune response can be enhanced by adding certain substances to the vaccines. These substances are called adjuvants, from the Latin adjuvare, which means to aid or help. Adjuvants can be used for various purposes. They can act to enhance the antigenicity of highly purified or recombinant antigens, they can reduce the amount of antigens or the number of immunizations needed for protective immunity, they can improve the efficacy of vaccines in newborns, elderly, or other immune-compromised persons, or they can act as antigen delivery systems for the uptake of antigens by mucosa. The chemical nature of adjuvants, their mechanism of action and their side effects are highly variable. Some of these side effects can be ascribed to an undesired stimulation of different mechanisms of the immune system, where others may reflect general adverse pharmacological reactions. There are many types of adjuvants. The most common adjuvants for human use are aluminum hydroxide, aluminum phosphate and calcium phosphate. There are also a number of adjuvants based on oil emulsions, products from bacterial (or their synthetic derivatives), endotoxins, fatty acids, paraffinic or vegetable oils, cholesterols, and aliphatic amines.
Aluminum salts, usually aluminum phosphate or aluminum hydroxide have been the most widely used adjuvants for human vaccines. Unfortunately, aluminum salts rarely induct cellular immune response and are generally relatively weak adjuvants. While the mechanism of action of aluminum salts is unknown, studies have suggested that they work by causing the formation of an antigen depot at the inoculation site from where the antigen is then slowly released. The immobilization of soluble antigens in the aluminum gel may also increase the duration of antigen interaction with the immune system. Other possible mechanisms of action involve complement, eosinophil and macrophage activation or an increased efficiency of antigen uptake by antigen presenting cells with a specific particulate matter size.
While aluminum salts have a relatively low rate of adverse effects, granulomas are common when the subcutaneous or intradermal injection route is used as opposed to intramuscular injection. Other limitations of aluminum adjuvants are increased IgE production, neurotoxicity and allergenicity. While under normal circumstances, small amounts of aluminum are excreted by the renal system, in situations of reduced kidney function, aluminum may accumulate in the body where it becomes highly toxic. In addition to aluminum salts, zirconium, iron and calcium salts have also been used to adsorb antigens. In particular, calcium salts have been used for diphtheria-tetanus-pertussis vaccines.
Adjuvant emulsions include oil in water or water in oil emulsions such as Freund's incomplete adjuvant (FIA), Montanide™, Adjuvant 65, and Lipovant™. These adjuvants work by forming a depot at the site of injection, enabling the meted release of antigenic material and the stimulation of antibody producing plasma cells. However, these adjuvants are have been deemed too toxic for widespread human prophylactic vaccine use and are usually reserved for those severe and/or terminal conditions such as cancer where there is a higher tolerance of side-effects.
Due to their potent immune-stimulatory capacity, bacteria-derived adjuvants are a major potential source of adjuvants. Lipopolysaccharide of Gram-negative bacteria or cell wall peptidoglycan enhances the immune response to co-administered antigenic material despite not being very antigenic themselves. This immune-stimulatory capacity works through the activation of toll-like receptors that activate the danger signals of the host immune system. Unfortunately, as killed or whole alive microorganisms these too are too toxic for widespread use in human prophylactic vaccines.
Problems with existing vaccines include at least the risks associated with adverse side effects, high cost, instability of the compound and/or its immunogenicity, the onset of an undesired disease state, and the spread of communicable potentially infectious agents. Thus, a clear need exists for a method of producing a vaccine for worldwide consumption with reduced risks of contamination, reliable stability and immunogenicity of the antigenic material, and an overall reduced quantity of antigenic material having the same or an equivalent efficacy as those currently available. In particular, there is demand for a safe and non-toxic adjuvant to stimulate cellular immunity.